This invention relates to modulating levels of enzymes and/or enzyme components relating to production of long chain poly-unsaturated fatty acids (PUFAs) in a microorganism or animal. The invention is exemplified by the production of arachidonic acid in yeast.
Two main families of poly-unsaturated fatty acids (PUFAs) are the xcfx893 fatty acids, exemplified by eicosapentaenoic acid (EPA), and the xcfx896 fatty acids, exemplified by arachidonic acid (ARA). PUFAs are important components of the plasma membrane of the cell, where they may be found in such forms as phospholipids and triglycerides. PUFAs are necessary for proper development, particularly in the developing infant brain, and for tissue formation and repair. PUFAs also serve as precursors to other molecules of importance in human beings and animals, including the prostacyclins, eicosanoids, leukotrienes and prostaglandins.
Four major long chain PUFAs of importance include docosahexaenoic acid (DHA) and EPA, which are primarily found in different types of fish oil, gamma-linolenic acid (GLA), which is found in the seeds of a number of plants, including evening primrose (Oenothera biennis), borage (Borago officinalis) and black currants (Ribes nigrum), and stearidonic acid (SDA), which is found in marine oils and plant seeds. Both GLA and another important long chain PUFA, arachidonic acid (ARA), are found in filamentous fungi. ARA can be purified from animal tissues including liver and adrenal gland. GLA, ARA, EPA and SDA are themselves, or are dietary precursors to, important long chain fatty acids involved in prostaglandin synthesis, in treatment of heart disease, and in development of brain tissue.
Several disorders respond to treatment with fatty acids. Supplementation with PUFAs has been shown to reduce the rate of restenosis after angioplasty. Fish oil supplements have been shown to improve symptoms of inflammation and rheumatoid arthritis, and PUFAs have been suggested as treatments for asthma and psoriasis. Evidence indicates that PUFAs may be involved in calcium metabolism, suggesting that PUFAs may be useful in the treatment or prevention of osteoporosis and of kidney or urinary tract stones.
PUFAs can be used in the treatment of cancer. Malignant cells have been shown to have altered fatty acid compositions; addition of fatty acids has been shown to slow their growth and cause cell death, and to increase their susceptibility to chemotherapeutic agents. GLA has been shown to cause reexpression on cancer cells of the E-cadherin cellular adhesion molecules, loss of which is associated with aggressive metastasis. Clinical testing of intravenous administration of the water soluble lithium salt of GLA to pancreatic cancer patients produced statistically significant increases in their survival. PUFA supplementation may also be usefull for treating cachexia associated with cancer.
PUFAs also can be used to treat diabetes (U.S. Pat. No. 4,826,877; Horrobin et al., Am. J. Clin. Nutr. Vol. 57 (Suppl.), 732S-737S). Altered fatty acid metabolism and composition has been demonstrated in diabetic animals. These alterations have been suggested to be involved in some of the long-term complications resulting from diabetes, including retinopathy, neuropathy, nephropathy and reproductive system damage. Primrose oil, which contains GLA, has been shown to prevent and reverse diabetic nerve damage.
Essential fatty acid deficiency has been suggested as being involved in eczema, and studies have shown beneficial effects on eczema from treatment with GLA. GLA has also been shown to reduce increases in blood pressure associated with stress, and to improve performance on arithmetic tests. GLA and DGLA have been shown to inhibit platelet aggregation, cause vasodilation, lower cholesterol levels and inhibit proliferation of vessel wall smooth muscle and fibrous tissue (Brenner et al., Adv. Exp. Med. Biol. Vol. 83, p. 85-101, 1976). Administration of GLA or DGLA, alone or in combination with EPA, has been shown to reduce or prevent gastrointestinal bleeding and other side effects caused by non-steroidal anti-inflammatory drugs (U.S. Pat. No. 4,666,701). GLA and DGLA have also been shown to prevent or treat endometriosis and premenstrual syndrome (U.S. Pat. No. 4,758,592) and to treat myalgic encephalomyelitis and chronic fatigue after viral infections (U.S. Pat. No. 5,116,871).
For DHA, a number of sources exist for commercial production including a variety of marine organisms, oils obtained from cold water marine fish, and egg yolk fractions. For ARA, microorganisms including the genera Mortierella, Entomophthora, Phytium and Porphyridium can be used for commercial production. Commercial sources of SDA include the genera Trichodesma and Echium. Commercial sources of GLA include evening prirnrose, black currants and borage. However, there are several disadvantages associated with commercial production of PUFAs from natural sources. Natural sources of PUFAs, such as animals and plants, tend to have highly heterogeneous oil compositions. The oils obtained from these sources therefore can require extensive purification to separate out one or more desired PUFAs or to produce an oil which is enriched in one or more PUFA. Natural sources also are subject to uncontrollable fluctuations in availability. Fish stocks may undergo natural variation or may be depleted by overfishing. Fish oils have unpleasant tastes and odors, which may be impossible to economically separate from the desired product, and can render such products unacceptable as food supplements. Animal oils, and particularly fish oils, can accumulate environmental pollutants. Weather and disease can cause fluctuation in yields from both fish and plant sources. Cropland available for production of alternate oil-producing crops is subject to competition from the steady expansion of human populations and the associated increased need for food production on the remaining arable land. Crops which do produce PUFAs, such as borage, have not been adapted to commercial growth and may not perform well in monoculture. Growth of such crops is thus not economically competitive where more profitable and better established crops can be grown. Large scale fermentation of organisms such as Mortierella is also expensive. Natural animal tissues contain low amounts of ARA and are difficult to process. Microorganisms such as Porphyridium and Mortierella are difficult to cultivate on a commercial scale.
Dietary supplements and pharmaceutical formulations containing PUFAs can retain the disadvantages of the PUFA source. Supplements such as fish oil capsules can contain low levels of the particular desired component and thus require large dosages. High dosages result in ingestion of high levels of undesired components, including contaminants. Unpleasant tastes and odors of the supplements can make such regimens undesirable, and may inhibit compliance by the patient. Care must be taken in providing fatty acid supplements, as overaddition may result in suppression of endogenous biosynthetic pathways and lead to competition with other necessary fatty acids in various lipid fractions in vivo, leading to undesirable results. For example, Eskimos having a diet high in xcfx893 fatty acids have an increased tendency to bleed (U.S. Pat. No. 4,874,603).
A number of enzymes are involved in PUFA biosynthesis. Linoleic acid (LA, 18:2 xcex949,12) is produced from oleic acid (18:1 xcex949) by a xcex9412-desaturase. GLA (18:3 xcex946,9,12) is produced from linoleic acid (LA, 18:2 xcex949,12) by a xcex946-desaturase. ARA (20:4 xcex945,8,11,14) production from dihomo-gamma-linolenic acid (DGLA, 20:3 xcex948,11,14) is catalyzed by a xcex945-desaturase. However, animals cannot desaturate beyond the xcex949 position and therefore cannot convert oleic acid (18:1 xcex949) into linoleic acid (18:2 xcex949,12). Likewise, xcex1-linolenic acid (ALA, 18:3 xcex949,12,15) cannot be synthesized by mammals. Other eukaryotes, including fungi and plants, have enzymes which desaturate at positions xcex9412 and xcex9415. The major poly-unsaturated fatty acids of animals therefore are either derived from diet and/or from desaturation and elongation of linoleic acid (18:2 xcex949,12) or xe2x88x9d-linolenic acid (18:3 xcex949,12,15). Therefore it is of interest to obtain genetic material involved in PUFA biosynthesis from species that naturally produce these fatty acids and to express the isolated material in a microbial or animal system which can be manipulated to provide production of commercial quantities of one or more PUFAs. Thus there is a need for fatty acid desaturases, genes encoding them, and recombinant methods of producing them. A need further exists for oils containing higher relative proportions of and/or enriched in specific PUFAs. A need also exists for reliable economical methods of producing specific PUFAs.
Relevant Literature
Production of gamma-linolenic acid by a xcex946-desaturase is described in U.S. Pat. No. 5,552,306. Production of 8,11-eicosadienoic acid using Mortierella alpina is disclosed in U.S. Pat. No. 5,376,541. Production of docosahexaenoic acid by dinoflagellates is described in U.S. Pat. No. 5,407,957. Cloning of a xcex946-palmitoyl-acyl carrier protein desaturase is described in PCT publication WO 96/13591 and U.S. Pat. No. 5,614,400. Cloning of a xcex946-desaturase from borage is described in PCT publication WO 96/21022. Cloning of xcex949-desaturases is described in the published patent applications PCT WO 91/13972, EP 0 550 162 A1, EP 0 561 569 A2, EP 0 644 263 A2, and EP 0 736 598 A1, and in U.S. Pat. No. 5,057,419. Cloning of xcex9412-desaturases from various organisms is described in PCT publication WO 94/11516 and U.S. Pat. No. 5,443,974. Cloning of xcex9415-desaturases from various organisms is described in PCT publication WO 93/11245. All publications and U.S. patents or applications referred to herein are hereby incorporated in their entirety by reference.
Novel compositions and methods are provided for preparation of poly-unsaturated long chain fatty acids or PUFAs. The compositions include nucleic acids encoding a xcex945-desaturase and/or polypeptides having xcex945-desaturase activity, the polypeptides, and probes for isolating and detecting the same. The methods involve growing a host microorganism or animal which contains and expresses one or more transgenes encoding a xcex945-desaturase and/or a polypeptide having xcex945-desaturase activity. Expression of the desaturase polypeptide provides for a relative increase in xcex945-desaturated PUFA, or metabolic progeny therefrom, as a result of altered concentrations of enzymes and substrates involved in PUFA biosynthesis. The invention finds use for example in the large scale production of PUFA containing oils which include, for example, ARA, EPA and/or DHA.
In a preferred embodiment, a nucleic acid sequence comprising a xcex945-desaturase depicted in FIG. 3A-E (SEQ ID NO 1), a polypeptide encoded by the nucleic acid, and a purified or isolated polypeptide depicted in FIG. 3A-E (SEQ ID NO: 2), and an isolated nucleic acid encoding the polypeptide of FIG. 3A-E (SEQ ID NO: 2) are provided. Another embodiment of the invention is an isolated nucleic acid sequence which encodes a polypeptide, wherein said polypeptide desaturates a fatty acid molecule at carbon 5 from the carboxyl end of the molecule. The nucleic acid is preferably derived from a eukaryotic cell, such as a fungal cell, or a fungal cell of the genus Mortierella, or of the genus/species Mortierella alpina. Also preferred is an isolated nucleic acid comprising a sequence which anneals to a nucleotide sequence depicted in FIG. 3A-3E (SEQ ID NO: 1), and a nucleic acid which encodes an amino acid sequence depicted in FIG. 3A-E (SEQ ID NO: 2). In particular, the nucleic acid encodes an amino acid sequence depicted in FIG. 3A-E (SEQ ID NO: 2) which is selected from the group consisting of amino acid residues 30-38, 41-44, 171-175, 203-212, and 387-394. In an additional embodiment, the invention provides an isolated or purified polypeptide which desaturates a fatty acid molecules at carbon 5 from the carboxyl end of the molecule. Also provided is an isolated nucleic acid sequence which hybridizes to a nucleotide sequence depicted in FIG. 3A-E (SEQ ID NO 1), an isolated nucleic acid sequence having at least about 50% identity to FIG. 3A-E (SEQ ID NO 1).
The present invention further includes a nucleic acid construct comprising a nucleotide sequence depicted in a FIG. 3A-E (SEQ ID NO: 1) linked to a heterologous nucleic acid; a nucleic acid construct comprising a nucleotide sequence depicted in a FIG. 3A-E (SEQ ID NO: 1) operably linked to a promoter; and a nucleic acid construct comprising a nucleotide sequence depicted in a FIG. 3A-E (SEQ ID NO: 1) operably linked to a promoter which is functional in a microbial cell. In a preferred embodiment, the microbial cell is a yeast cell, and the nucleotide sequence is derived from a fungus, such as a fungus of the genus Mortierella, particularly a fungus of the species Mortierella alpina. 
In another embodiment of the invention, a nucleic acid construct is provided which comprises a nucleotide sequence which encodes a polypeptide comprising an amino acid sequence which corresponds to or is complementary to an amino acid sequence depicted in FIG. 3A-E (SEQ ID NO: 2), wherein the nucleotide sequence is operably linked to a promoter which is functional in a host cell, and wherein the nucleotide sequence encodes a polypeptide which desaturates a fatty acid molecule at carbon 5 from the carboxyl end of a fatty acid molecule. Additionally, provided by the invention is a nucleic acid construct comprising a nucleotide sequence which encodes a functionally active xcex945-desaturase, where the desaturase includes an amino acid sequence which corresponds to or is complementary to all of or a portion of an amino acid sequence depicted in a FIG. 3A-E (SEQ ID NO: 2), wherein the nucleotide sequence is operably linked to a promoter functional in a host cell.
The invention also includes a host cell comprising a nucleic construct of the invention. In a preferred embodiment, a recombinant host cell is provided which comprises at least one copy of a DNA sequence which encodes a functionally active Mortierella alpina fatty acid desaturase having an amino acid sequence as depicted in FIG. 3A-E (SEQ ID NO: 2), wherein the cell or an ancestor of the cell was transformed with a vector comprising said DNA sequence, and wherein the DNA sequence is operably linked to a promoter. The host cell is either eukaryotic or prokaryotic. Preferred eukaryotic host cells are those selected from the group consisting of a mammalian cell, an insect cell, a fungal cell, and an algae cell. Preferred mammalian cells include an avian cell, a fungal cell such as a yeast, and a marine algae cell. Preferred prokaryotic cells include those selected from the group consisting of a bacteria, a cyanobacteria, cells which contain a bacteriophage, and/or a virus. The DNA sequence of the recombinant host cell preferably contains promoter which is functional in the host cell.
The host cells of the invention which contain the DNA sequences of the invention are enriched for fatty acids, such as 20:3 fatty acids. In a preferred embodiment, the host cells are enriched for 20:4 fatty acids as compared to an untransformed host cell which is devoid of said DNA sequence, and/or enriched for 20:5 fatty acids compared to an untransformed host cell which is devoid of said DNA sequence. In yet another preferred embodiment, the invention provides a recombinant host cell which comprises a fatty acid selected from the group consisting of a dihomo-xcex3-linoleic acid, n-6 eicosatrienoic acid, 20:3n-6 acid and 20:3 (8,11,14) acid.
The present invention also includes method for production of arachidonic acid in a microbial cell culture, where the method comprises growing a microbial cell culture having a plurality of microbial cells which contain one or more nucleic acids encoding a polypeptide which converts dihomo-xcex3-linoleic acid to arachidonic acid, wherein the nucleic acid is operably linked to a promoter, under conditions whereby said one or more nucleic acids are expressed, whereby arachidonic acid is produced in the microbial cell culture. In several preferred embodiments of the invention, the polypeptide is an enzyme which desaturates a fatty acid molecule at carbon 5 from the carboxyl end of the fatty acid molecule; the nucleic acid is derived from a Mortierella sp.; and the substrate for said polypeptide is exogenously supplied. The microbial cells used in the methods can be either eukaryotic cells or prokaryotic cells. The preferred eukaryotic cells are those selected from the group consisting of a mammalian cell, an insect cell, a fungal cell, and an algae cell. Preferred mammalian cells include an avian cell, a preferred fungal cell is a yeast, and the preferred algae cell is a marine algae cell. The preferred prokaryotic cells include those selected from the group consisting of a bacteria, a cyanobacteria, cells which contain a bacteriophage, and/or a virus. The nucleic acid sequence encoding the polypeptide of the microbial cell preferably contains a promoter which is functional in the host cell which optionally is an inducible promoter for example by components of the culture broth. The preferred microbial cells used in the methods are yeast cells, such as Saccharomyces cells.
In another embodiment of the invention, a recombinant yeast cell is provided which converts greater than about 5% of 20:3 fatty acid substrate to a 20:4 fatty acid substrate
Also provided is an oil comprising one or more PUFA. The amount of said one or more PUFAs is approximately 0.3-30% arachidonic acid (ARA), approximately 0.2-30% dihomo-xcex3-linoleic acid (DGLA), and approximately 0.2-30% xcex3-linoleic acid (GLA). A preferred oil of the invention is one in which the ratio of ARA:DGLA:GLA is approximately 1.0:19.0:30 to 6.0:1.0:0.2. Another preferred embodiment of the invention is a pharmaceutical composition comprising the oils in a pharmaceutically acceptable carrier. Further provided is a nutritional composition comprising the oils of the invention. The nutritional compositions of the invention preferably are administered to a mammalian host parenterally or internally. A preferred composition of the invention for internal consumption is an infant formula. In a preferred embodiment, the nutritional compositions of the invention are in a liquid form or a solid form.
The present invention also includes a method for desaturating a fatty acid, where the method comprises culturing a recombinant microbial cell of the invention under conditions suitable for expression of a polypeptide encoded by the nucleic acid, wherein the host cell further comprises a fatty acid substrate of the polypeptide. In a preferred embodiment, a fatty acid desaturated by the methods is provided, including an oil comprising the fatty acid.